Surface enhanced spectroscopy-active composite nanoparticles

ABSTRACT

Metal nanoparticles associated with a spectroscopy-active (e.g., Raman-active) analyte and surrounded by an encapsulant are useful as sensitive optical tags detectable by surface-enhanced spectroscopy (e.g., surface-enhanced Raman spectroscopy).

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/680,782, filed Oct. 6, 2000, entitled “SurfaceEnhanced Spectroscopy-Active Composite Nanoparticles,” incorporatedherein by reference, which claims priority to U.S. ProvisionalApplication No. 60/157,931, filed Oct. 6, 1999, entitled “Glass CoatedSurface Enhanced Raman Scattering Tags,” and U.S. ProvisionalApplication No. 60/190,395, filed Mar. 17, 2000, entitled “GANSParticles.”

FIELD OF THE INVENTION

[0002] This invention relates generally to submicron-sized tags orlabels that can be covalently or non-covalently affixed to entities ofinterest for the purpose of quantification, location, identification, ortracking. More particularly, it relates to surface enhancedspectroscopy-active composite nanoparticles, methods of manufacture ofthe particles, and uses of the particles.

BACKGROUND OF THE INVENTION

[0003] When light is directed onto a molecule, the vast majority of theincident photons are elastically scattered without a change infrequency. This is termed Rayleigh scattering. However, the energy ofsome of the incident photons (approximately 1 in every 10⁷ incidentphotons) is coupled into distinct vibrational modes of the molecule'sbonds. Such coupling causes some of the incident light to beinelastically scattered by the molecule with a range of frequencies thatdiffer from the range of the incident light. This is termed the Ramaneffect. By plotting the frequency of such inelastically scattered lightagainst its intensity, the unique Raman spectrum of the molecule underobservation is obtained. Analysis of the Raman spectrum of an unknownsample can yield information about the sample's molecular composition.

[0004] The incident illumination for Raman spectroscopy, usuallyprovided by a laser, can be concentrated to a small spot if thespectroscope is built with the configuration of a microscope. Since theRaman signal scales linearly with laser power, light intensity at thesample can be very high in order to optimize sensitivity of theinstrument. Moreover, because the Raman response of a molecule occursessentially instantaneously (without any long-lived highly energeticintermediate states), photobleaching of the Raman-active molecule—evenby this high intensity light—is impossible. This places Ramanspectroscopy in stark contrast to fluorescence spectroscopy, in whichphotobleaching dramatically limits many applications.

[0005] The Raman effect can be significantly enhanced by bringing theRaman-active molecule(s) close (≦50 Å) to a structured metal surface;this field decays exponentially away from the surface. Bringingmolecules in close proximity to metal surfaces is typically achievedthrough adsorption of the Raman-active molecule onto suitably roughenedgold, silver or copper or other free electron metals. Surfaceenhancement of the Raman activity is observed with metal colloidalparticles, metal films on dielectric substrates, and with metal particlearrays. The mechanism by which this surface-enhanced Raman scattering(SERS) occurs is not well understood, but is thought to result from acombination of (i) surface plasmon resonances in the metal that enhancethe local intensity of the light, and; (ii) formation and subsequenttransitions of charge-transfer complexes between the metal surface andthe Raman-active molecule.

[0006] SERS allows detection of molecules attached to the surface of asingle gold or silver nanoparticle. A Raman enhancing metal nanoparticlethat has associated or bound to it a Raman-active molecule(s) can haveutility as an optical tag. For example, the tag can be used inimmunoassays when conjugated to an antibody against a target molecule ofinterest. If the target of interest is immobilized on a solid support,then the interaction between a single target molecule and a singlenanoparticle-bound antibody can be detected by searching for theRaman-active molecule's unique Raman spectrum. Furthermore, because asingle Raman spectrum (from 100 to 3500 cm⁻¹) can detect many differentRaman-active molecules, SERS-active nanoparticles may be used inmultiplexed assay formats.

[0007] SERS-active nanoparticles with adsorbed Raman-active moleculesoffer the potential for unprecedented sensitivity, stability, andmultiplexing functionality when used as optical tags in chemical assays.However, metal nanoparticles present formidable practical problems whenused in such assays. They are exceedingly sensitive to aggregation inaqueous solution; once aggregated, it is not possible to re-dispersethem. In addition, the chemical compositions of some Raman-activemolecules are incompatible with the chemistries used to attach othermolecules (such as proteins) to metal nanoparticles. This restricts thechoices of Raman-active molecules, attachment chemistries, and othermolecules to be attached to the metal nanoparticle.

SUMMARY OF THE INVENTION

[0008] One embodiment of the present invention provides surface-enhancedspectroscopy (SES)-active composite nanoparticles, such as SERS-activecomposite nanoparticles (SACNs). Such nanoparticles each contain aSES-active metal nanoparticle; a submonolayer, monolayer, or multilayerof spectroscopy-active species associated with or in close proximity(e.g., adsorbed) to the metal surface; and an encapsulating shell madeof a polymer, glass, or any other dielectric material. This places thespectroscopy-active molecule (alternately referred to herein as the“analyte,” not to be confused with the species in solution that isultimately being quantified) at the interface between the metalnanoparticle and the encapsulant.

[0009] In some embodiments, the encapsulant is glass. The resultingglass-coated analyte-loaded nanoparticles (GANs) retain the activity ofthe spectroscopy-active analyte, but lightly sequester this activityfrom the exterior surface of the nanoparticle. Thus, in the case ofsurface-enhanced Raman scattering (SERS), the resulting GAN exhibitsSERS activity, but the Raman-active analyte is located at the interfacebetween the metal nanoparticle and the encapsulant.

[0010] The analyte molecule can be chosen to exhibit extremely simpleRaman spectra, because there is no need for the species to absorbvisible light. This, in turn, allows multiple composite nanoparticles,each with different analyte molecules, to be fabricated such that theRaman spectrum of each analyte can be distinguished in a mixture ofdifferent types of particles.

[0011] Surface-enhanced spectroscopy (SES)-active compositenanoparticles are easily handled and stored. They are also aggregationresistant, stabilized against decomposition of the analyte in solventand air, chemically inert, and can be centrifuged and redispersedwithout loss of SERS activity. SACNs may be provided as a dispersion insuitable solvent for storage or association with an object or molecule.

[0012] In one embodiment, the encapsulant may be readily derivatized bystandard techniques. This allows the particles to be conjugated tomolecules (including biomolecules such as proteins and nucleic acids) orto solid supports without interfering with the Raman activity of theparticles. Unlike metal nanoparticles, SACNs can be evaporated todryness and then completely redispersed in solvent. Using the techniquesprovided herein, it is possible to fabricate particles that areindividually detectable using SERS.

[0013] In an alternative embodiment, SACNs are attached to, mixed with,or otherwise associated with objects for tracking, identification, orauthentication purposes. Each type of particle or group of particletypes, as defined by the Raman spectrum of the encapsulated analyte,represents a particular piece of information. Tagged objects areverified by acquiring their Raman spectrum. Any liquid, solid, orgranular material can be tagged with a SACN. By associating an objectwith more than one different SACN type, a large number of distinct SACNgroups can be obtained.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1A is a transmission electron micrograph of GANs with 35 nmAu cores and 40 nm glass shells.

[0015]FIG. 1B is a transmission electron micrograph of GANs with 35 nmAu cores and 16 nm glass shells.

[0016]FIG. 2 is a transmission electron micrograph of 35 nm Au, 8 nmglass GANs following centrifugation in a 50% glycerol solution.

[0017]FIGS. 3A and 3B are plots of absorbance versus wavelength, fordifferent etch times, and absorbance versus etch time at a singlewavelength, respectively, for a particle having a 35 nm Au core and 8 nmglass shell.

[0018]FIG. 4 shows Raman spectra of GANs with a 40 nm Au coreencapsulated in 4 mm of glass, with (trace A) and without (trace B) theRaman-active analyte, trans-1,2-bis(4-pyridyl)ethylene (BPE).

[0019]FIG. 5 shows Raman spectra of a suspension of GANs with 40 nm Aucoated with BPE and 4 nm glass encapsulant (Trace A); supernatant from afirst centrifugation of the GANs (Trace B); and supernatant from asecond centrifugation of the GANs (Trace C).

[0020]FIG. 6 shows Raman spectra of GANs (80 nm Aucore/2-mercaptopyridine/20 nm glass) and of a 50 mM solution of2-mercaptopyridine absorbed onto a conventional three-layer SERSsubstrate.

[0021]FIG. 7 shows Raman spectra of the following four types (“flavors”)of GANs particles: (A) GANs tagged with furonitrile; (B) GANs taggedwith furonitrile (66%) and cyanoacetic acid (33%); (C) GANs tagged withfuronitrile (33%) and cyanoacetic acid (66%); and (D) GANs tagged withcyanoacetic acid.

[0022]FIG. 8 shows Raman spectra of GANs (40 nm Au core/4 nm glass) (a)tagged with BPE; (b) tagged with imidazole; or (c) untagged.

[0023]FIGS. 9A and 9B show Raman spectra of GANs containing BPE andpara-nitroso-N,N′-dimethylaniline (p-NDMA) in ethanol spotted onto whiteand yellow paper, respectively.

DETAILED DESCRIPTION OF EMBODIMENTS

[0024] Various embodiments of the present invention are directed tosurface-enhanced spectroscopy-active composite nanoparticles (SACNs),including surface-enhanced Raman spectroscopy (SERS)-active compositenanoparticles. Other embodiments provide methods of manufacture of theparticles and methods of use of the particles. These submicron-sizedtags or labels can be covalently or non-covalently affixed to or mixedwith entities of interest, ranging in size from molecular tomacroscopic, for the purpose of quantification, location,identification, or tracking.

[0025] The SACNs provided by embodiments of the present invention areuniquely identifiable nanoparticles. They can be used in virtually anysituation in which it is necessary to label molecules or objects with anoptical tag. Biomolecules can be conjugated readily to the exterior ofSACNs by standard techniques, thereby allowing the particles to functionas optical tags in biological assays. SACNs can be used in virtually anyassay that uses an optical tag, such as a fluorescent label. However, asoptical tags, SACNs have several distinct advantages over fluorescentlabels. These advantages include vastly more sensitive detection,chemical uniformity, and the absolute resistance of the SERS activity tophotobleaching or photodegradation. A further benefit of using SACNs asoptical tags is the ease with which individual SACNs having differentSERS activities may be resolved from one another. At least twentydifferent SACNs are resolvable from one another using a simple Ramanspectroscope. This enables multiplexed assays to be performed using apanel of different SACNs, each having a unique and distinguishable SERSactivity. It also provides for, when SACNs are formed into groups, avery large number of unique SACN combinations with which objects can belabeled.

[0026] A surface-enhanced spectroscopy-active composite nanoparticle(SACN) contains a surface-enhanced spectroscopy (SES)-active (e.g.,SERS-active) metal nanoparticle that has attached to or associated withits surface one or more spectroscopy-active (e.g., Raman-active)molecules (alternately referred to herein as “analytes”). This complexof Raman-enhancing metal and analyte is then coated or encapsulated byan encapsulant. A SACN typically has a diameter of less than 200 nm or,alternatively, less than 100 nm. Each SACN is identified by the distinctRaman spectrum of its Raman-active analyte. Note that a metalnanoparticle is referred to as “surface-enhanced spectroscopy active”because it acts to enhance the spectroscopic signal of the associatedanalyte; it is the signal of the analyte, which is inherentlyspectroscopy active, that is being measured.

[0027] SACNs can be produced by growing or otherwise placing a shell ofa suitable encapsulant over a SERS-active metal nanoparticle core withassociated Raman-active analyte. The metal nanoparticle core can be, forexample, a gold or silver sphere of between about 20 nm and about 200 nmin diameter. In another embodiment, the metal nanoparticle has adiameter of between about 40 nm and about 100 nm. In an alternativeembodiment, the metal nanoparticle is an oblate or prolate metalspheroid. For SERS using red incident light (˜633 nm), a suitable SERSresponse can be obtained with 63 nm diameter gold nanoparticles, butother particle diameters can also be employed. Typically, a SACNcontains only one metal nanoparticle, but more than one particle can beencapsulated together if desired. In a collection or plurality of SACNs,some may have one metal nanoparticle and some more than one metalnanoparticle. In this collection of particles, at least some of thedetected Raman signal originates from the particle or particlescontaining only one metal nanoparticle. Because the Raman signalintensity is a substantially linear function of the number ofRaman-active analytes, limiting a group of SACNs to a known number ofmetal nanoparticles and adsorbed analyte molecules allows estimation ofthe number of particles from the signal intensity of a group ofparticles.

[0028] The metal nanoparticles can contain any SES-active metal, i.e.,any metallic substance for which chemical enhancement, electromagneticenhancement, or both, is known in the art. For example, the metalnanoparticles can contain Au, Ag, or Cu. Other suitable metals include,but are not limited to, Na, K, Cr, Al, or Li. The metal particles canalso contain alloys of metals. In one embodiment, the metal nanoparticleconsists of a core (of pure metal or an alloy) overlaid with at leastone metal shell. In this case, the composition of the outer layer can bechosen to maximize the intensity of the Raman signal from the analyte.

[0029] Metal nanoparticles of the desired size can be grown as metalcolloids by a number of techniques well known in the art. For example,chemical or photochemical reduction of metal ions in solution using anynumber of reducing agents has been described. Likewise, nanoparticlesyntheses have been carried out in constrained volumes, e.g. inside avesicle. Nanoparticles can also be made via electrical discharge insolution. Dozens of other methods have been described, dating back tothe mid-1800's.

[0030] The Raman-active analyte can be any molecular species having ameasurable SERS spectrum. Whether or not a spectrum is measurable maydepend upon the instrument with which the spectrum is acquired and theamount of adsorbed analyte. In general, however, a measurable spectrumis one that is detectable in the presence of the metal nanoparticle andencapsulant and can be recognized as characteristic of the particularanalyte. Typically, the maximum intensity of the SERS spectrum of theanalyte is substantially greater than that of the particle without theanalyte (i.e., the metal nanoparticle and encapsulant). Aromaticmolecules often have measurable Raman spectra. Examples of aromaticmolecules suitable for use as analytes in embodiments of SACNs include,but are not limited to, trans-1,2-bis(4-pyridyl)ethylene (BPE),pyridine, 2-mercaptopyridine, furonitrile, imidazole, andpara-nitroso-N,N′-dimethylaniline (p-NDMA).

[0031] Those skilled in the art will recognize that there is a greatdeal of latitude in the composition of an analyte that yields a distinctRaman spectrum. For example, in some embodiments, the analyte is not amolecule: it can be a positively or negatively charged ion (e.g., Na⁺ orCN⁻). If the analyte is a molecule, it can be neutral, positivelycharged, negatively charged, or amphoteric. The analyte can be a solid,liquid or gas. Non-molecular species such as metals, oxides, sulfides,etc. can serve as the Raman-active species. Any species or collection ofspecies that gives rise to a unique Raman spectrum, whether solid,liquid, gas, or a combination thereof, can serve as the analyte.Examples easily number in the many millions and include but are notlimited to Hg, dimethylformamide, HCl, H₂O, CN⁻, polypyrrole,hemoglobin, oligonucleotides, charcoal, carbon, sulfur, rust,polyacrylamide, citric acid, and diamond. In the case of diamond, theunique phonon mode of the particle can be used. For hemoglobin, only theporphyrin prosthetic group exhibits significant Raman activity; thus,complex substances can be used as the analyte if only part of themolecular or atomic complexity is present in the Raman spectrum.

[0032] The analyte can also be a polymer to which multiple Raman-activemoieties are attached. In this case, differentiable SACNs contain thesame polymer serving as the analyte, but the polymers have differentattached moieties yielding different Raman spectra. The polymer backbonedoes not itself contribute to the acquired Raman spectrum. In oneembodiment, the polymer is a linear chain containing amine groups towhich Raman-active entities are attached. Alternatively, the polymer canbe a dendrimer, a branched polymer with a tightly controlled tree-likestructure, with each branch terminating in a Raman-active species. Asuitable dendrimer structure has four generations of branchesterminating in approximately 45 Raman-active entities.

[0033] Note that SACNs that give rise to unique Raman spectra can beconsidered different even if the analyte is essentially the same. Forexample, the Raman spectrum of a cationic polymer charge compensated byanions can change depending on the choice of counter ion. A panel ofdifferentiable SACNs can be formed using this polymer as a component ofthe analyte; each unique SACN has the polymer charge-compensated by adifferent anion, thereby endowing each SACN with a unique Ramanspectrum. In addition, a given analyte may have different Raman shiftson different SERS-active layers, and differentiable SACNs can be formedusing the same analyte sandwiched between layers of different metals.For example, p-NDMA has different Raman shifts on gold and silversurfaces. Alternatively, one or more bands in the Raman spectrum of ananalyte may be dependent on the density of the analyte in the SACN.SACNs formed with different densities of the same analyte are thereforedifferentiable from one another.

[0034] As will be apparent to one skilled in the art, characteristics ofsome suitable Raman-active analytes are (i) strong Raman activity, whichminimizes the number of molecules needed to provide a given signalstrength; and (ii) simple Raman spectrum, which allows a large number ofunique SACNs to be distinguished when used simultaneously.

[0035] The Raman-active analyte can form a sub-monolayer, a completemonolayer, or a multilayer assembly on the surface of the metalnanoparticle core. A Raman-active analyte can be a single species ofRaman-active molecule, a mixture of different species of Raman-activemolecules, or a mixture of Raman-active molecules and molecules withoutmeasurable Raman activity.

[0036] Typically, the encapsulant does not measurably alter the SERSactivity of the metal nanoparticle or the Raman spectrum of the analyte.In an alternative embodiment, however, the encapsulant can have ameasurable effect without adding significant complexity to the Ramanspectrum. The encapsulant can be readily modified in order to attachmolecules, including biomolecules, to its exterior surface. Suitableencapsulants include, but are not limited to, glass, polymers, metals,metal oxides (such as TiO₂ and SnO₂), and metal sulfides. Theencapsulation is carried out after, or during, adsorption to the corenanoparticle of the Raman-active analyte that is to provide the Ramanactivity of the SACN. In this way, the Raman-active analyte issequestered from the surrounding solvent. Such a configuration providesthe metal nanoparticle core with stable SERS activity. Alternatively, itmay not be necessary to sequester the analyte completely, in which casethe encapsulant does not completely surround the metal nanoparticle andanalyte.

[0037] The thickness of the encapsulant can be easily varied dependingon the physical properties required of the SACN. For example, coatingsthat are too thick—on the order of 1 micron or more—might precludeobtaining intense Raman spectra. Coatings too thin might lead tointerference in the Raman spectrum of the analyte by molecules on theencapsulant surface. Raman scattering intensity decreases exponentiallywith distance between analyte and nanoparticle surface; beyond 2 nm, theenhancing effect is negligible. An encapsulant that is at least thisthick prevents interference in the spectrum from molecules on theoutside of the SACN. Physical properties such as sedimentationcoefficient will clearly be affected by the thickness of encapsulant. Ingeneral, the thicker the encapsulant, the more effective thesequestration of the Raman-active analyte(s) on the metal nanoparticlecore from the surrounding solvent. One suitable thickness range of theencapsulant is between about 1 nm and about 40 nm. Alternatively, theencapsulant can be between about 5 nm and about 15 nm thick. Anothersuitable thickness range is between about 10 nm and about 20 nm.

[0038] In one embodiment of the invention, the encapsulant is glass(e.g., SiO_(x)). To encapsulate in glass, the metal nanoparticle coresare preferably treated first with a glass primer (that is, a materialthat can lead to growth of a uniform coating of glass, or can improveadhesion of the glass coat to the particle, or both). Glass is thengrown over the metal nanoparticle by standard techniques well known inthe art. The resulting SACNs are referred to as glass analyte-loadednanoparticles (GANs). For GANs, a suitable glass thickness ranges fromabout 1 nm to about 40 nm or, alternatively, between about 10 nm andabout 20 nm. In one embodiment, the GAN contains a 60 nm diameter goldparticle encapsulated by a 16 nm thick shell of glass. In an alternativeembodiment, the encapsulant is TiO₂, which is chemically similar to SiO₂and commonly used in many industries.

[0039] It may be desirable to separate true SACNs from free particles ofencapsulant that were not nucleated around a metal nanoparticle. Suchseparation improves the SERS activity of the nanoparticle preparationbecause free encapsulant particles are not SERS active. For example,GANs can be separated from free glass particles by size-exclusioncentrifugation in 50% glycerol.

[0040] Note that glass and many other materials contain functionalgroups amenable to molecular attachment. For example, immersion of glassin base allows covalent attachment of alkyl trichlorosilanes or alkyltrialkoxysilanes, with additional functionality available on the end ofthe alkyl group. Thus, glass surfaces can be modified with all forms ofbiomolecules and biomolecular superstructures including cells, as wellas oxides, metals, polymers, etc. Likewise, surfaces of glass can bemodified with well-organized monomolecular layers. In short, glasscoatings support essentially any and all forms of chemicalfunctionalization (derivatization). This is equally true for manydifferent forms of encapsulant, so that SACNs can be affixed to anyspecies with chemically reactive functionality. All chemical functionalgroups are reactive under certain conditions. There is thus nolimitation to the species that can be immobilized on the encapsulantsurface.

[0041] The optimization of the dimensions of the SACNs is readilyaccomplished by one skilled in the art. Accordingly, one might alter thecomposition of the particle, or its size and shape, in accordance withthe invention to optimize the intensity of the Raman signal. Indeed, itis known that core-shell nanoparticles (i.e. Au/AuS) support SERS andhave very different optical properties compared to pure metalnanoparticles. Likewise, it is known that SERS from prolate spheroids isenhanced relative to spheres with the same major axis. It is furtherknown that single particle enhancements are stronglywavelength-dependent. Thus, one might “tune” the particle size, shape,and composition to achieve maximum signal for a given excitationwavelength.

[0042] One embodiment of the present invention contemplates theformation of a panel of at least 20 different SACNs, each having aunique SERS spectrum. This panel is referred to herein as a collectionof distinguishable particles. Because the Raman bands of many moleculesare extremely narrow (for example, CN⁻ is less than 1 nm at FWHM), it ispossible to synthesize a panel of SACNs, each containing a Raman analytethat is spaced 20 wavenumbers away in the spectrum from its closestneighbor. For example, a GAN with ¹³CN as the analyte is easilydistinguished from a GAN with ¹²CN as the analyte, and as well easilydistinguishable from one with C¹⁵N. In this way, it is possible to form540 distinct and easily resolvable peaks in a single Raman spectrum at633 nm from 300 to 3000 cm⁻¹ using a spectrograph to spread the photonsand a CCD camera as a detector. In general, Raman-active analytes can beused that have isotopic compositions distinct from naturally abundantspecies. For example, as described above, ¹³CN is completely resolvablefrom any natural ¹²CN that may be present in the background. Of course,those skilled in the art will recognize that combinations of isotopes aswell as ratios of isotopes can be equally effectively used to identifyunique SACNs.

[0043] Raman experiments with GANs or other SACNs can also be carriedout with visible or near-IR irradiation, make use of Raman bands from100 cm⁻¹ to 5000 cm⁻¹, employ any form of monochromator or spectrometerto spatially or temporally resolve photons, or employ any form of photondetector. This arrangement facilitates the synthesis of panels of atleast 10 resolvable SACNs, and provides ample bandwidth for literallyhundreds of panels of SACNs.

[0044] Although the SERS activity of each population of SACNs in thepanel is unique, the other properties of the SACNs are kept uniformacross the panel. Because the SERS activity of each SACN is sequesteredfrom the surrounding milieu by the encapsulant, individual populationsdo not have different solvent or storage requirements. Also, each SACNhas the same exterior shell, simplifying the choice of chemistry eitherfor attachment of molecules to the SACNs or attachment of the SACNs tosolid supports.

[0045] While the examples above have focused on Raman scattering, and inparticular surface-enhanced Raman scattering as the detection mechanism,a number of analogous methods can apply equally well and are includedwithin the scope of the present invention. For example, one can employ aresonantly-excited analyte, thus making the technique surface-enhancedresonance Raman scattering (SERRS). One could also take advantage ofexisting methods of surface-enhanced infrared absorption spectra (SEIRA)from nanoscale roughened surfaces. Likewise, surface-enhanced hyperRamanscattering (SEHRS) also occurs at nanoscale roughened metal surfaces,and this technique as well as its resonant analogue SEHRRS can beemployed. Note that for a given molecule, with either 3N-5 or 3N-6unique vibrations, where N is the number of atoms, all vibrations can befound in either the Raman, hyperRaman, or infrared spectrum. Indeed,identification of certain SACNs could rest on a combination of opticalinterrogation methods, including SERS, SERRS, SEIRA, SEHRS and SEHRRS.

[0046] Note also that a significant amount of (Rayleigh) lightscattering is known to occur from particles with dimensions at least{fraction (1/10)} the exciting wavelength, thus creating the possibilitythat Rayleigh or hyperRayleigh scattering could be used inidentification of SACNs. Moreover, combinations of elastic scattering(e.g. Rayleigh), inelastic scattering (e.g. Raman), and absorption (e.g.IR) could be used to identify particles.

[0047] Use of SACNs

[0048] The SACNs provided by embodiments of the present invention can beused in virtually any application in which a detectable tag or label isrequired. In some embodiments, SACNs are used in biological and chemicalassays as replacements for standard fluorescent tags. Indeed, SACNspossess a number of characteristics that make them far superior to priorart optical tags based on fluorophores. For example, assays usingfluorophore detection are commonly hampered by the presence ofautofluorescence and other background effects. In addition, many assaysrequire use of a number of different fluorophores; differentfluorophores commonly require different attachment chemistries and havedifferent environmental requirements and sensitivities. Particularlynoteworthy is the quenching of fluorescent activity that is observedwhen some fluorophores are conjugated to proteins. Finally, irreversiblephotodegradation resulting from the creation of a triplet or singletexcited state, followed by a non-reversible chemical reaction thatpermanently eliminates the excited state, places a severe limitation onthe sensitivity of detection. By contrast, SACNs cannot be photobleachedor photodegraded, they have uniform chemical and physical properties,and they can be readily resolved from the background. Perhaps mostimportantly, SACN detection is significantly more sensitive thanfluorophore detection. Indeed, it is possible to tag a single moleculewith a single SACN, and then detect the presence of that molecule usingRaman spectroscopy. Such simple single molecule resolution is withoutparallel in the fluorophore detection art.

[0049] An example of a biological assay in which SACNs can be used asoptical tags is the sandwich immunoassay. In sandwich assays, a targetto be detected is captured by a solid surface. An antibody (or otherligand) to the same target is attached to a SACN, and then contactedwith the solid support. The presence of the SACN SERS signal at thesolid support indicates the presence of the antigen. In general, SACNscan be conjugated to any molecule that is used to detect the presence ofa specific target in an assay.

[0050] In a specifically contemplated embodiment, SACNs are conjugatedto nucleic acid molecules. In this way, they can be used in virtuallyany assay known in the art that detects specific nucleic acid sequencesusing optically-tagged nucleic acid probes.

[0051] SACNs are especially suitable for multiplexed chemical assays inwhich the identity of SACNs encodes the identity of the target of theassay. Prior art multiplexed assays that use fluorophores to encodetarget identity are subject to a number of severe constraints imposed bythe physical and chemical properties of the fluorophores. Specifically,different fluorophores have different excitation maxima, so coincidentexcitation of multiple fluorescent tags is not possible. Moreover,fluorescence emission occurs in broad spectral bands, so the bands fromone fluorophore often overlap with those of another. As a result,resolving even three different fluorescence activities requiressophisticated optics to separate and then detect the individual emissionwavelengths. Because of these problems, multiplexed assays that usefluorophores rely on positional information to reveal target identity.Often, multiplexed assays with fluorophores use a solid support on whichligands are arranged in defined positions. The location of fluorophoresignal reveals the identity of the target; the size of the fluorophoresignal at that location indicates the amount of the target. However, thesynthesis of solid supports with reagents localized at specificpositions is expensive and time-consuming. Also, there are limits on thenumber of features that may be defined on a single surface.

[0052] By contrast, the SACNs of the present invention offer remarkablespectral diversity and resolvability. As a result, SACNs can be used inmultiplexed assays to yield quantitative and qualitative informationwithout requiring the position-specific localization of reagents. EachSACN coupled to a target-specific reagent can encode the identity ofthat specific target, and the intensity of a particular Raman signalreveals the quantity of that target. For example, in the sandwichimmunoassays described above, the identity of targets captured on thesolid support can be determined by using a different flavor of SACN foreach target.

[0053] Although SACNs are perfectly suited for use in multiplexingapplications, they need not be used to encode identity in this manner.They can be used simply as replacements for fluorophores in multiplexedassays in which reagents are localized to specific positions on solidsupports. When used in this way, the SACNs offer vastly more sensitivetarget detection than fluorophores.

[0054] In other embodiments, SACNs serve as tags for labeling objects ormaterials, e.g., for anti-counterfeiting or authentication purposes, orfor encoding the history of an object moving through a manufacturingprocess or supply chain. In these applications, one or more SACNs areassociated with an object or material and later “read” by Ramanspectroscopy to determine the identity of the particle or particles andobtain information about the tagged object. The acquired spectrum can becompared to a reference spectrum or to a spectrum of the particlesacquired before they were associated with the object. If necessary,suitable corrections can be made to account for background emission fromthe object. Authentication can occur at any desired point during thelifetime of the object, e.g., upon receipt of a manufactured object by aretailer or upon sale of an antique object.

[0055] Each SACN or group of SACNs, with its unique Raman spectrum,corresponds to or represents a particular piece of information. Any typeof information can be represented by a SACN, depending upon theapplication. For example, a SACN or group of SACNs can represent anindividual object such as an item of sports memorabilia, a work of art,an automobile, or the item's owner or manufacturer; a class of objects,such as a particular formulation of pharmaceutical product; or a step ofa manufacturing process. The information represented by a particularRaman spectrum or SACN type can be stored in a database, computer file,paper record, or other desired format.

[0056] The small, robust, non-toxic, and easily-attachable nature ofSACNs allows their use for tagging virtually any desired object. Thetracked object can be made of solid, liquid, or gas phase material orany combination of phases. The material can be a discrete solid object,such as a container, pill, or piece of jewelry, or a continuous orgranular material, such as paint, ink, fuel, or extended piece of, e.g.,textile, paper, or plastic, in which case the particles are typicallydistributed throughout the material.

[0057] Examples of specific materials or objects that can be tagged withSACNs include, but are not limited to:

[0058] Packaging, including adhesives, paper, plastics, labels, andseals

[0059] Agrochemicals, seeds, and crops

[0060] Artwork

[0061] Computer chips

[0062] Cosmetics and perfumes

[0063] Compact disks (CDs), digital video disks (DVDs), and videotapes

[0064] Documents, money, and other paper products (e.g., labels,passports, stock certificates)

[0065] Inks, paints, and dyes

[0066] Electronic devices

[0067] Explosives

[0068] Food and beverages, tobacco

[0069] Textiles, clothing, footwear, designer products, and apparellabels

[0070] Polymers

[0071] Hazardous waste

[0072] Movie props and memorabilia, sports memorabilia and apparel

[0073] Manufacturing parts

[0074] Petroleum, fuel, lubricants, oil

[0075] Pharmaceuticals and vaccines

[0076] Particles can be associated with the material in any way thatmaintains their association at least until the particles are read.Depending upon the material to be tagged, the particles can beincorporated during production or associated with a finished product.Because they are so small, the particles are unlikely to have adetrimental effect on either the manufacturing process or the finishedproduct. The particles can be associated with or attached to thematerial via any chemical or physical means. For example, particles canbe mixed with and distributed throughout a liquid-based substance suchas paint, oil, or ink and then applied to a surface. They can be woundwithin fibers of a textile, paper, or other fibrous or woven product, ortrapped between layers of a multi-layer label. The particles can beincorporated during production of a polymeric or slurried material andbound during polymerization or drying of the material. Additionally, thesurfaces of the particles can be chemically derivatized with functionalgroups of any desired characteristic, as described above, for covalentor non-covalent attachment to the material. When the particles areapplied to a finished product, they can be applied manually by, e.g., apipette, or automatically by a pipette, spray nozzle, or the like.Particles can be applied in solution in a suitable solvent (e.g.,ethanol), which then evaporates.

[0077] SACNs have a number of inherent properties that are advantageousfor tagging and tracking applications. They offer a very large number ofpossible codes. For example, if a panel of SACNs is constructed with 20distinguishable Raman spectra, and an object is labeled with two SACNs,there are 20*19/2=190 different codes. If the number of particles perobject is increased to 5, there are 15,504 possible codes. Ten particlesper object yields 1.1×10⁶ different codes. A more sophisticatedmonochromator increases the number of distinguishable Raman spectra to,e.g., 50, greatly increasing the number of possible codes.

[0078] SACNs can be identified using a conventional Raman spectrometer.In fact, one benefit of using SACNs is the versatility of excitationsources and detection instrumentation that can be employed for Ramanspectroscopy. Visible or near-IR lasers of varying sizes andconfigurations can be used to generate Raman spectra. Portable,handheld, and briefcase-sized instruments are commonplace. At the sametime, more sophisticated monochromators with greater spectral resolvingpower allow an increase in the number of unique taggants that can beemployed within a given spectral region. For example, the capability todistinguish between two Raman peaks whose maxima differ by only 3 cm⁻¹is routine.

[0079] Typically, if a suitable waveguide (e.g., optical fiber) isprovided for transmitting light to and from the object, the excitationsource and detector can be physically remote from the object beingverified. This allows SACNs to be used in locations in which it isdifficult to place conventional light sources or detectors. The natureof Raman scattering and laser-based monochromatic excitation is suchthat it is not necessary to place the excitation source in closeproximity to the Raman-active species.

[0080] Another characteristic of SACNs is that the measurement of theirRaman spectra need to strictly be confined to “line of sight” detection,as with, e.g., fluorescent tags. Thus their spectrum can be acquiredwithout removing the particles from the tagged object, provided that thematerial is partially transparent to both the excitation wavelength andthe Raman photon. For example, water has negligible Raman activity anddoes not absorb visible radiation, allowing SACNs in water to bedetected. SACNs can also be detected when embedded in, e.g., clearplastic, paper, or certain inks.

[0081] SACNs also allow for quantitative verification, because the Ramansignal intensity is an approximately linear function of the number ofanalyte molecules. For standardized particles (uniform analytedistribution), the measured signal intensity reflects the number ordensity of particles. If the particles are added at a knownconcentration, the measured signal intensity can be used to detectundesired dilution of liquid or granular materials.

[0082] SACNs are chemically and biologically inert, and a glass coatinggives the particles charge, flow, and other physical properties similarto those of SiO₂ particles commonly used as excipients in pills,vitamins, and a wide variety of other materials. A TiO₂ coating on SACNsallow them to be used in the very large number of materials thatcurrently contain TiO₂, such as papers, paints, textiles, and apparel.

[0083] Because of their submicron size, SACNs can be added to fluidswithout changing the fluid properties significantly or affecting thefluid handling equipment. For example, the particles can flow throughnarrow tubes and be expelled out nozzles without clogging lines ororifices.

[0084] SACNs are also non-toxic and can be ingested safely by humans andother animals. This enables their tagging of pharmaceutical products,food products, and beverages (e.g., wine). Particles are comparable insize to the excipients normally used as vehicles for drugs.

[0085] The following examples are offered by way of illustration and notby way of limitation.

WORKING EXAMPLES Working Example 1 Synthesis of Glass-coatedAnalyte-loaded Nanoparticles (GANs)

[0086] Materials: Water used for all preparations was 18.2 MΩ, distilledthrough a Barnstead nanopure system. Snake skin dialysis tubing, 3,500MWCO, was purchased from Pierce. 3-aminopropyltrimethoxysilane (APTMS),3-mercaptotrimethoxysilane (MPTMS), and3-mercaptopropylmethyldimethoxysilane (MPMDMS) were obtained from UnitedChemical. HAuCl₄.3H₂O, trisodium citrate dihydrate, sodium hydroxide,trans-1,2-bis(4-pyridyl)ethylene (BPE), pyridine, 2-mercaptopyridine,sodium silicate, tetraethyl orthosilicate (TEOS), and ammonia wereobtained from Sigma-Aldrich. BPE was recrystallized several times beforeuse. Dowex cation exchange resin (16-40 mesh) was obtained from J. T.Baker. Pure ethyl alcohol (EtOH) was purchased from Pharmco.

[0087] Colloid preparation: 12-nm colloidal Au (nearly spherical, with astandard deviation less than 2 nm) was prepared from HAuCl₄.3H₂O reducedby citrate as described in Grabar et al, Analytical Chemistry 67:735-743(1995), incorporated herein by reference in its entirety.

[0088] Colloid>12 nm was prepared as follows: 3 ml of 12 mM HAuCl₄ wasadded for every 97 ml of H₂O. The solution was then brought to a boilunder vigorous stirring and 1 ml of 12-nm Au colloid as a seed and 0.5ml of 1% sodium citrate per 100 ml of HAuCl₄ solution was added andboiled for 10 minutes. The size of the resulting particles wasdetermined by transmission electron microscopy using Gatan or NIH Imagesoftware. Finally, the citrate ions surrounding the Au colloid wereremoved with dialysis, 7 exchanges of at least 4 hours each.

[0089] GANs preparation: All reactions were performed in plasticErlenmeyer flasks. Any amount of colloid could be used in a preparationand the subsequent reactants added in appropriate amounts based on thesurface area and concentration of the Au colloid.

[0090] A typical experiment used 25 ml of dialyzed, 50-nm, 0.17 nM Aucolloid. The pH of the colloid was adjusted from 5 to 7 with theaddition of 50 μL of 0.1 M NaOH. The colloid was rendered vitreophilicwith the addition 125 μL of 0.5 mM MPTMS (or APTMS, or MPMDMS). After 15minutes of magnetic stirring, 167 μL of a 0.5 mM solution of the Ramantag (BPE, pyridine, or 2-mercaptopyridine) was added. During another 15minute period of stirring, a 0.54% solution of active silica wasprepared by mixing 1 g of sodium silicate with 50 ml of 3 M NaOH andlowering the pH to 10 with cation exchange resin. One ml of the activesilica was added and the resulting solution was approximately pH 9. Thesolution remained stirring for 15 minutes and then was allowed to stand.

[0091] After a 24 hour period, 100 ml of EtOH was added to the solutionto proceed with silica growth via the method described in Stöber et al,J. Colloid Interface Sci. 26: 62 (1968), incorporated herein byreference in its entirety. Growth of ˜4 nm of additional glass shell wasaccomplished with the addition of 15 μL of TEOS and 125 μL of ammonia.The reaction was stirred for 15 minutes and then allowed to stand for atleast 12 hours. The addition of TEOS and ammonia was continued until thedesired shell thickness was obtained.

Working Example 2 Transmission Electron Microscopy of GANs

[0092] Transmission electron microscopy (TEM) images were taken ofpreparations of GANs; these TEM images illustrate the uniformity of GANspreparations. FIG. 1A shows GANs containing 35 nm Au cores with 40 nmglass. FIG. 1B shows 60 nm Au cores with 16 nm glass. FIG. 2 illustrates35 nm Au, 8 nm glass GANs following centrifugation through a 50%glycerol solution.

Working Example 3 Demonstration of the Sequestration of the Metal Corefrom Solvent

[0093] For GANs to function in diverse chemical environments, it isnecessary that the Raman-active analyte be sequestered from thesurrounding solvent. To demonstrate this sequestration, one may look atdiffusion rates through the glass network. This is done by monitoringthe rate at which aqua regia (3 HCl:1 HNO₃) is able to etch out the Aucore of a GAN. FIG. 3 demonstrates one such experiment for a batch ofGANs particles with a 35 nm Au core, and 8 nm shell of glass. To 500 μlof ≈0.17 nM GANs was added 200 μl of an etch solution (50 μl HNO₃ and150 μl HCl). The absorbance of the solution was measured (λ_(max) 546nm) at various times after addition of the etch solution. Etching of thegold core results in a decrease in the absorbance; this is plotted inFIG. 3A (the time after the addition of the etch solution is indicated).The rate of Au etching is shown in FIG. 3B as a plot of absorbanceversus time in etch solution (right). Additional studies performed bythe inventors have shown that etching of a Au core by aqua regia doesnot occur with a 20 nm glass shell over a four hour time period.

Working Example 4 SERS Spectra of GANs Particles

[0094] GANs containing a 40 nm Au core coated withtrans-1,2-bis(4-pyridyl)ethylene (BPE) encapsulated in 4 nm of glasswere synthesized and examined by Raman spectroscopy. The Raman spectrumobtained using 20 mW of 632.8 nm excitation, with a 3 mm lens and 30second integration is plotted in FIG. 4. Trace A on the graph shows thecharacteristic BPE Raman signal; trace B shows the Raman signal from thesame particles without the BPE analyte. It can be seen that the GANswithout the BPE analyte give essentially no Raman signal.

Working Example 5 Confinement of the Raman-active Analyte to the MetalCore of GANs by Glass Encapsulation

[0095]FIG. 5 shows the Raman spectrum of a suspension of GANs comprising40 nm Au coated with trans-1,2-bis(4-pyridyl)ethylene (BPE)/4 nm glass(Trace A); supernatant from a first centrifugation of the GANs (TraceB); and supernatant from a second centrifugation of the GANs (Trace C).It can be seen that the BPE signal does not leave the GAN during eachcentrifugation step, indicating that the BPE has adhered to the Au coreand is tightly sequestered there by glass encapsulation.

Working Example 6 Comparison of SERS Spectra of Raman-active Analytes onGANs with Other SERS Substrates

[0096] GANs (80 nm Au core/20 nm glass) containing 2-mercaptopyridine asthe Raman-active analyte were analyzed by Raman spectroscopy using 25 mWof 632.8 nm excitation with a 3 mm lens and 60 seconds of integration.The Raman spectrum of the GANs preparation was then compared with theRaman spectrum obtained when a 50 mM solution of 2-mercaptopyridine isabsorbed onto a conventional three-layer SERS substrate (25 mW 632.8 nmexcitation, 3 mm lens, 30-seconds integration). FIG. 6 shows the twoRaman spectra. It can be seen that the two spectra have identicalfeatures and intensities, illustrating that the metal nanoparticles ofthe GANs are effective SERS substrates.

Working Example 7 SERS Spectra of GANs with Mixtures of Raman-activeAnalytes

[0097] SERS spectra of the following four flavors of GANs particles wereobtained using 26 mW of 632.8 nm excitation, a 3-mm lens, and 30-secondintegration: (A) GANs tagged with furonitrile; (B) GANs tagged withfuronitrile (66%) and cyanoacetic acid (33%); (B) GANs tagged withfuronitrile (33%) and cyanoacetic acid (66%); and (D) GANs tagged withcyanoacetic acid. The percentages indicated are the relativeconcentrations of each compound in the tagging solution added. FIG. 7shows that the furonitrile and cyanoacteic acid have relatively the samesignal intensity and have similar spectral profiles. The fact that thespectra of B and C are very similar to the spectrum of D indicates thatcyanoacetic acid has a better affinity for the Au nanoparticle thanfuronitrile.

Working Example 8 SERS Spectra of GANs Tagged with Imidazole (IM) andtrans-4, 4′-bis(pyridyl)ethylene (BPE)

[0098] GANs (40 nm Au core/4 nm glass) were tagged with either (a)trans-1,2-bis(4-pyridyl)ethylene (BPE-GANs) or (b) imidazole (IM-GANs).SERS spectra of these Raman-active analytes are shown in FIG. 8, alongwith the SERS spectrum of untagged GANs (c) of the same dimensions.BPE-GANs and IM-GANs both show the characteristic Raman bands of theirrespective Raman-active analytes; untagged GANs do not show these bands.

Working Example 9 Tagging Paper with GANs

[0099] GANs were prepared from 12 nm-diameter gold particles coated withtrans-1,2bis-(4-pyridyl)ethylene (BPE) orpara-nitroso-N,N′-dimethylaniline (p-NDMA) as described in WorkingExample 1. Encapsulation was completed with a single addition of TEOSand ammonia. Particles were stored in ethanol as prepared (at aconcentration of approximately 1 nM).

[0100] The particle solutions were mixed and three spots each ofapproximately 10-20 μl of the resulting solution were pipetted ontoyellow and white sheets of conventional paper. After evaporation ofethanol, spots were visible on both sides of the paper, indicating thatthe particles had penetrated the sheets.

[0101] Raman spectra of the spots were acquired using approximately 20mW of 633 nm excitation with a 3 mm lens and 30 seconds of integration.FIG. 9A shows a representative spectrum on white paper and FIG. 9B onyellow paper. Both the yellow and white paper displayed high levels ofbackground fluorescence. However, characteristic peaks of BPE at about1200, 1610, and 1640 cm⁻¹ were detectable over the background signal inboth cases.

[0102] It should be noted that the foregoing description is onlyillustrative of the invention. Various alternatives and modificationscan be devised by those skilled in the art without departing from theinvention. Accordingly, the present invention is intended to embrace allsuch alternatives, modifications and variances which fall within thescope of the disclosed invention.

What is claimed is:
 1. A particle comprising exactly onesurface-enhanced spectroscopy (SES)-active metal nanoparticle, aspectroscopy-active analyte associated with said SES-active metalnanoparticle, and an encapsulant surrounding said SES-active metalnanoparticle and said spectroscopy-active analyte, wherein saidspectroscopy-active analyte has a measurable SES spectrum.
 2. Theparticle of claim 1, wherein said metal nanoparticle comprises a metalchosen from at least one of Cu, Na, Al, and Cr.
 3. The particle of claim1, wherein said metal nanoparticle comprises Au.
 4. The particle ofclaim 1, wherein said metal nanoparticle comprises Ag.
 5. The particleof claim 1 wherein said metal nanoparticle has a diameter of less thanabout 200 nm.
 6. The particle of claim 5 wherein said metal nanoparticlehas a diameter of between about 40 nm and about 100 nm.
 7. The particleof claim 1 wherein said encapsulant has a thickness of between about 1nm and about 40 nm.
 8. The particle of claim 7 wherein said encapsulanthas a thickness of between about 5 nm and about 15 nm.
 9. The particleof claim 1, wherein said metal nanoparticle comprises a core overlaidwith at least one metal shell, and wherein said core and at least one ofsaid metal shells each comprise a metal chosen from at least one of Au,Ag, Cu, Na, Al, and Cr.
 10. The particle of claim 1 wherein said metalnanoparticle comprises an alloy of at least two metals chosen from Au,Ag, Cu, Na, Al, and Cr.
 11. The particle of claim 1, wherein saidspectroscopy-active analyte forms a submonolayer coating on said metalnanoparticle.
 12. The particle of claim 1, wherein saidspectroscopy-active analyte forms a monolayer coating on said metalnanoparticle.
 13. The particle of claim 1, wherein saidspectroscopy-active analyte forms a multilayer coating on said metalnanoparticle.
 14. The particle of claim 1, wherein said encapsulantcomprises at least one material chosen from glass, polymers, metals,metal oxides, and metal sulfides.
 15. The particle of claim 1, whereinsaid encapsulant comprises at least two materials chosen from glasses,polymers, metals, metal oxides, and metal sulfides.
 16. The particle ofclaim 1 wherein said encapsulant comprises glass oxide (SiO_(x)). 17.The particle of claim 1 wherein said encapsulant comprises TiO₂.
 18. Theparticle of claim 1 wherein said surface-enhanced spectroscopy is chosenfrom at least one of SERS, SERRS, SEHRRS, and SEIRA.
 19. The particle ofclaim 1, wherein said spectroscopy-active analyte is an aromaticanalyte.
 20. The particle of claim 19, wherein said aromatic analyte ischosen from at least one of BPE, p-NDMA, pyridine, 2-mercaptopyridine,furonitrile, and imidazole.
 21. The particle of claim 1, wherein saidspectroscopy-active analyte has a SES spectrum with a maximum intensitysubstantially greater than that of said particle without saidspectroscopy-active analyte.
 22. A particle comprising exactly onesurface-enhanced spectroscopy (SES)-active metal nanoparticle, aspectroscopy-active analyte associated with said SES-active metalnanoparticle, and a network of glass oxide (SiO_(x)) surrounding saidSES-active metal nanoparticle and said spectroscopy-active analyte,wherein said spectroscopy-active analyte has a measurable SES spectrum.23. The particle of claim 22 wherein said surface-enhanced spectroscopyis chosen from at least one of SERS, SERRS, SEHRRS, and SEIRA.
 24. Aparticle comprising exactly one gold nanoparticle having a diameter ofbetween about 40 nm and about 100 nm, a spectroscopy-active analyteassociated with said gold nanoparticle, and glass having a thickness ofbetween about 5 nm and about 15 nm surrounding said gold nanoparticleand said spectroscopy-active analyte, wherein said spectroscopy-activeanalyte has a measurable surface-enhanced spectroscopy spectrum.
 25. Theparticle of claim 24 wherein said surface-enhanced spectroscopy isselected from the group consisting of SERS, SERRS, SEHRRS, and SEIRA.26. A particle comprising exactly one metal nanoparticle, an aromaticanalyte adsorbed on the surface of said metal nanoparticle, and anencapsulant surrounding said metal nanoparticle and said aromaticanalyte.
 27. The particle of claim 26, wherein said aromatic analyte ischosen from at least one of BPE, p-NDMA, pyridine, 2-mercaptopyridine,furonitrile, and imidazole.
 28. The particle of claim 26, wherein saidmetal nanoparticle comprises a metal chosen from at least one of Au, Ag,Cu, Na, Al, and Cr.
 29. The particle of claim 26, wherein said metalnanoparticle has a diameter of less than about 200 nm.
 30. The particleof claim 29, wherein said metal nanoparticle has a diameter of betweenabout 40 nm and about 100 nm.
 31. The particle of claim 26, wherein saidencapsulant has a thickness of between about 1 nm and about 40 nm.
 32. Acollection of distinguishable particles, each particle comprising: asurface-enhanced spectroscopy (SES)-active metal nanoparticle; aspectroscopy-active analyte associated with said SES-active metalnanoparticle; and an encapsulant surrounding said SES-active metalnanoparticle and said spectroscopy-active analyte; wherein saiddistinguishable particles comprise spectroscopy-active analytes havingdistinguishable spectra.
 33. The collection of claim 32, wherein saidspectroscopy is Raman spectroscopy.
 34. The collection of claim 32,wherein said collection comprises at least about 20 distinguishableparticles.
 35. A dispersion comprising a solvent and at least oneparticle, said particle comprising: a surface-enhanced spectroscopy(SES)-active metal nanoparticle, a spectroscopy-active analyteassociated with said SES-active metal nanoparticle, and an encapsulantsurrounding said SES-active metal nanoparticle and saidspectroscopy-active analyte, wherein said spectroscopy-active analytehas a measurable SES spectrum.
 36. A plurality of particles, eachcomprising: a surface-enhanced spectroscopy (SES)-active metalnanoparticle, a spectroscopy-active analyte associated with saidSES-active metal nanoparticle, said spectroscopy-active analyte having ameasurable SES spectrum, and an encapsulant surrounding said SES-activemetal nanoparticle and said spectroscopy-active analyte, wherein atleast some of a spectrum acquired from said plurality of particlesoriginates from a particle comprising exactly one SES-active metalnanoparticle.
 37. A method of manufacturing particles comprising: a)associating a spectroscopy-active analyte with a surface-enhancedspectroscopy (SES)-active metal nanoparticle; and b) coating saidanalyte-associated metal nanoparticle with an encapsulant; wherein saidspectroscopy-active analyte has a measurable SES spectrum.
 38. A methodof tagging a material, comprising associating with said material atleast one particle comprising: a surface-enhanced spectroscopy(SES)-active metal nanoparticle, a spectroscopy-active analyte, and anencapsulant surrounding said SES-active metal nanoparticle and saidspectroscopy-active analyte.
 39. The method of claim 38, wherein saidspectroscopy is Raman spectroscopy.
 40. The method of claim 38, whereinsaid material comprises a liquid.
 41. The method of claim 40, whereinsaid material is chosen from at least one of ink, paint, and oil. 42.The method of claim 38, wherein said material is chosen from at leastone of paper, textiles, polymers, and pharmaceuticals.
 43. The method ofclaim 38, wherein associating with said material at least one particlecomprises associating with said material at least two particles, eachparticle having a distinct Raman spectrum.
 44. The method of claim 38,further comprising acquiring a SES spectrum of said material and saidassociated particle.